Excited-State Barrier Controls E → Z Photoisomerization in p-Hydroxycinnamate Biochromophores

Molecules based on the deprotonated p-hydroxycinnamate moiety are widespread in nature, including serving as UV filters in the leaves of plants and as the biochromophore in photoactive yellow protein. The photophysical behavior of these chromophores is centered around a rapid E → Z photoisomerization by passage through a conical intersection seam. Here, we use photoisomerization and photodissociation action spectroscopies with deprotonated 4-hydroxybenzal acetone (pCK–) to characterize a wavelength-dependent bifurcation between electron autodetachment (spontaneous ejection of an electron from the S1 state because it is situated in the detachment continuum) and E → Z photoisomerization. While autodetachment occurs across the entire S1(ππ*) band (370–480 nm), E → Z photoisomerization occurs only over a blue portion of the band (370–430 nm). No E → Z photoisomerization is observed when the ketone functional group in pCK– is replaced with an ester or carboxylic acid. The wavelength-dependent bifurcation is consistent with potential energy surface calculations showing that a barrier separates the Franck–Condon region from the E → Z isomerizing conical intersection. The barrier height, which is substantially higher in the gas phase than in solution, depends on the functional group and governs whether E → Z photoisomerization occurs more rapidly than autodetachment.


Photodissociation action spectroscopy
The photodissociation action spectrum for p CK − was recorded in a modied DMS-MS device capable of irradiating target anions in the third quadrupole region of a triple quadrupole mass spectrometer with tunable wavelength laser light. Complete details of the DMS-MS apparatus are given in Refs 13; for a description of the instrument modications enabling photodissociation action spectroscopy see Ref. 4. The target anion was electrosprayed from acetonitrile (≈1 µg mL −1 ) with 0.1% NH 4 OH (to assist deprotonation). Ions were carried through the planar DMS cell by dry nitrogen gas. Ionograms showed evidence for only one isomer, consistent with the tandem ion mobility experiments detailed in the next section. For the photodissociation measurements, the compensation voltage applied to the DMS cell was xed to transmit ions from individual ion populations into a triple-quadrupole mass spectrometer (Q1Q3). Ions were mass selected in Q1 (m/z 161), transmitted through Q2 and accumulated in Q3 for ≈10 ms. Trapped ions were irradiated for ≈500 ms with light from a tunable optical parametric oscillator (OPO, Horizon II, Continuum, loosely focused, 25 mJ pulse −1 ) pumped by a pulsed Nd:YAG laser (10 Hz, Surelite, Continuum). The parent ions and any photofragment ions were ejected from the ion trap following a mass-selective axial ejection toward a channeltron ion detector. 5 Ion fragmentation eciencies were normalized with respect to ion count, OPO uence, and photon number. Photodissociation was dominated by two fragments, corresponding to loss of CH 3 and C 2 H 4 O.

Photoisomerizaiton action spectroscopy
Photoisomerization and prompt photodetachment of p CK − was investigated in a custom tandem ion mobility spectrometer (IMS-IMS) coupled with a quadrupole mass lter (QMF). 6,7 Briey, p CK − was produced through electrospray ionisation of a ≈10 µmol L −1 solution (Sigma-Aldrich, >99 %) dissolved in methanol (voltage -3 kV, ow rate ≈10 µL min −1 ). Electrosprayed ions were transferred via a heated capillary into a RF ion funnel (IF1), which radially gathered and conned the ions. An ion gate (IG1) at the end of IF1 injected ≈100 µs packets of ions at 40 Hz into the rst IMS drift region (IMS1) where they were propelled by an electric eld (44 V cm −1 ) through pure N 2 or CO 2 buer gas, N 2 seeded with ≈1% SF 6 and ≈1% propan-2-ol, or CO 2 seeded with ≈1% propan-2-ol (see Supporting Information) at a pressure of ≈6 Torr. The isomers become separated spatially and temporally because more extended ions (E isomers) have dierent collision cross-sections with the buer gas than the Z isomers. 8 After traversing the drift regions (IMS1 + IMS2), a second ion funnel collected the ions and introduced them into a dierentially pumped octupole ion guide and quadrupole mass lter that mass-selected the ions before they reached the ion detector. The detector was connected to a multichannel scaler that produced a histogram of ion counts against arrival time, t, corresponding to an arrival time distribution (ATD). In all presented ATDs, t =0 corresponds to the opening of IG1. The mobility resolution, t/∆t, for singly-charged anions is typically 80 90. 9 For the action spectroscopy measurements, packets of ions with similar collision cross-sections were selected using a Bradbury-Nielsen ion gate after IMS1 (IG2, ≈100 µs opening time). Immediately after gating, the mobility-selected ions were excited with a pulse of light from an optical parametric oscillator (OPO, EKSPLA NT342B). Any change in the ions' collision cross-section due to photoisomerization was manifested as a shift in arrival time following passage through a second IMS drift region (IMS2). The OPO was operated at 20 Hz, half the rate of ion injection, allowing accumulation of light-on and light-o ATDs. The dierence between the light-on and light-o ATDs reected the photoresponse (photoaction ATD). Action spectra were derived by integrating the photoaction ATD signal, and normalizing with respect to light pulse uence and total laser-o signal at each wavelength. The action spectroscopy measurements were performed with a light pulse uence of < 0.5 mJ cm −2 to minimize multiphoton absorption and sequential photoisomerizations.

Time-resolved uorescence upconversion
Time-resolved uorescence upconversion measurements were performed using an instrument that has been previously detailed. 10 Briey, a CW Nd:YVO4 laser drives a Kerr lens mode-locked Ti:Sapphire (Ti:S) oscillator generating ≈800 nm light in ≈20 fs pulses at 80 MHz. The second harmonic (400 nm, ≈11 mW) was generated by focussing the fundamental (≈840 mW) light into a 50 µm thickness barium borate crystal (BBO, type I) using a 150 mm focal length concave mirror. The fundamental and second harmonic wavelengths were separated with a dichroic mirror. The 400 nm and 800 nm light pulses were delayed relative to each other using a computer-controlled motorised delay stage (0.1 µm resolution). The use of chirped mirrors minimized temporal broadening. The 400 nm pump pulse was focussed onto the sample held in either a static 2 mm path length quartz cell or 2 mm path length quartz ow cell using a concave mirror. Resulting uorescence was captured and focussed by a microscope objective (15x magnication) through a CG455 Schott lter into a 100 µm thickness BBO crystal (type I) and frequency mixed with 800 nm light. The unconverted signal was passed through a UG11 Schott lter into a monochromator (Photon Technology International Model 101, resolution 2 nm/mm) and low-noise photomultiplier (PMT, Hamamatsu R585). PMT signal is connected to a computer-interfaced Stanford Research Systems photon counter (SR400). The cross correlation of the instrument has been characterised through Raman scattering in heptane to be ≈55 fs.
The uorescence quantum yield for p CK − at T = 300 K has been estimated at less than 10 −3 . 11 The ow rate in the ow cell was systematically increased until measured up-converted lifetimes no longer changed with increasing ow rate.

S3
Theoretical methods Electronic structure calculations Electronic structure calculations were performed using the Gaussian 16.B01, Firey 8.2.0, and ORCA 5.0.3 software packages. 1214 Geometrical optimizations, vibrational frequencies, and isomerization transition state searches were performed at the ωB97X-D/aug-cc-pVTZ level of theory, 15,16 followed by single-point energy calculations at the DLPNO-CCSD(T)/aug-cc-pVTZ level of theory. 17 Vertical detachment energies (VDEs) were calculated at the EOM-IP-CCSD/aug-cc-pVDZ level of theory, based on good performance in a recent study. 9 Collision cross-sections were calculated using MOBCAL with the trajectory method parametrised for N 2 buer gas. 18,19 Input charge distributions were computed at the ωB97X-D/aug-cc-pVDZ level of theory with the Merz-Singh-Kollman scheme constrained to reproduce the electric dipole moment. 20 Sucient trajectories were computed to give standard deviations of ±1 Å 2 for the calculated values.
For the potential energy surface calculations, S 0 and S 1 state equilibrium geometries, TS ‡ , and conical intersection geometries were optimized at the state-specic CASSCF(10,9)/aug-cc-pVDZ level of theory. Single-point energy calculations were performed at the XMCQDPT2(12,11)/augcc-pVDZ level of theory. The active spaces chosen for the geometry optimizations and single-point energy calculations are a balance between accuracy and computation cost following the earlier study by Boggio-Pasqua and Groenhof. 21

S6
Action spectroscopy of pCK − in CO 2 buer gas In addition to the action spectra recorded in N 2 buer gas (seeded with SF 6 and propan-2-ol) shown in the paper, action spectra were recorded in CO 2 buer gas ( Figure S3). The purpose of these measurements was two-fold: (i) to investigate if CO 2 buer gas provided better separation of E and Z isomers compared with N 2 buer gas, 22 and (ii) to investigate if the action spectra change appearance with buer gas identity because CO 2 is more ecient at collisional energy transfer/quenching than N 2 .
The photoaction ATDs for p CK − in pure CO 2 and CO 2 seeded with propan-2-ol are shown in Figure S3a and b, respectively. For pure CO 2 buer gas, a small Z -isomer signal is observed on the Figure S3: Ion mobility action spectroscopy of p CK − : (a) light-o (black) and photoaction (blue) ATDs in CO 2 buer gas, (b) light-o (black) and photoaction (blue) ATDs in CO 2 buer gas seeded with ≈1% propan-2-ol, (c) action spectra recorded in CO 2 buer gas and (d) action spectra recorded in CO 2 buer gas seeded with ≈1% propan-2-ol. The photoaction spectra show the appearance (positive) and bleaching (negative) of signals. The quadrupole mass lter was set to the m/z of the parent ion. fast side of the light-o ATD peak. For CO 2 seeded with ≈1% propan-2-ol, the Z -isomer signal is observed on the slower side of the light-o ATD peak and is slightly better resolved than in pure CO 2 buer gas. Photodepletion and E-Z photoisomerization action spectra (normalized) are shown in Figure S3c and d, respectively. These sets of spectra closely resemble each other and parallel the action spectra reported in the paper, thus suggesting that that any dierences in collisional energy quenching eciency between N 2 and CO 2 buer gas does not alter the appearance of the action spectra.
S8 pCK − ·propan-2-ol complexes The addition of a small quantity of a dopant or`mobility modier' to the buer gas used in ion mobility is a common practice to assist in separation of isomers with similar collision cross-sections. 23 Briey, the collision cross-section, Ω, can be approximated as the sum of two contributions: where Ω s is the small impact parameter term accounting for hard-sphere type interactions and Ω l is the large impact parameter term accounting for glancing collisions. The latter of these terms is sensitive to long-range interactions (e.g. dipole-quadrupole and hydrogen bonding).
To help understand the larger collision cross-section for (Z )-pCK − compared with (E )-pCK − in N 2 + propan-2-ol (or CO 2 + propan-2-ol) buer gas, we computed minimum energy structures and complex binding energies for the species shown in Figure S4. Based on DLPNO-CCSD(T)/augcc-pVDZ energies, the Z1-complex was ≈8 kJ mol −1 more stable (to dissociating) than the corresponding E1 complex, consistent with an increased Ω l term. However, it is important to note that, because the photoisomerization and photodetachment (or photodepletion) spectra recorded in buer gases with propan-2-ol dopant closely resemble those recorded in pure buer gas, we conclude that any ion-molecule complexes are transitory and do no alter the action spectra. Figure S4: Calculated structures (ωB97X-D/aug-cc-pVDZ) for p CK − ·propan-2-ol complexes.

Solution spectroscopy
Fluorescence excitation and emission spectra for p CK − in ethanol at T = 300 K and T = 77 K are shown in Figure S6a. There is a substantial decrease in Stokes shift from 4143±20 cm −1 (T = 300 K) to 2243±20 cm −1 (T = 77 K). The large Stokes shift at T = 300 K is because of nuclear and solvent relaxation from the Franck-Condon geometry to the uorescing geometry. In contrast, the same extent of nuclear reorganization does not occur in the glassy matrix at T = 77 K. Absorption and uorescence emission spectra for p CK − in waterethylene-glycol mixtures are shown in Figure S7. Spectral properties of p CK − in the various solvents at T = 300 K are summarized in Table S1.

Fluorescence upconversion
To explore the dependence of uorescence lifetime on emission wavelength, up-converted signals from p CK − in water were monitored at a series of wavelengths ( Figure S8). These data show the uorescence lifetime to increase at longer monitoring wavelength. This is presumably because the lifetimes (close to 1 ps) are in competition with vibrational energy relaxation and nuclear reorganisation processes. 24 Following the discussion in Ref. 11, these lifetimes are comparable with the ≈880 fs long component of solvent dynamics in water. 25 For consistency in measurements across solvents, uorescence lifetimes were recorded for up-converted signals corresponding to the wavelength of maximum response in the emission spectrum. Lifetimes extracted from ts to the experimental data are summarized in Table S2. When dierences exist between static and ow cell uorescence lifetimes, ow cell lifetimes are assumed more reliable due to establishment of a partial photostationary state in the static cell. Fits to the upconversion data (e.g. see Figure S10) assumed either a Gaussian-like instrument response function convoluted with a single-exponential decay (S 1 τ 0 − →S 0 ) or a double-exponential decay scheme S 1 − →S 0 , where S 1,rel is the relaxed excited state after solvent reorganization. 25 In most cases, τ 1 is limited by the cross correlation of the experiment. The lifetimes and t residuals indicate that the double-exponential t is most benecial for the more viscous, less polar solvents due to slower solvent reorganization. These data improve on earlier upconversion experiments on p CK − with ≈500 fs time resolution. 11 For example, for p CK − in water, Espagne et al. 11 quoted τ 1 = 0.5 ps and τ 2 = 1.3 ps, to be compared with τ 1 = 0.055 ps (limited by cross correlation) and τ 2 = 1.17 ps (1.13 ps in a static cell) in the current work.
In water, there is only a 0.04 ps dierence between uorescence lifetimes measured in the ow and static cell, which although small, is beyond tted uncertainty. This suggests that either there is only a small amount of Z-isomer formed, or the E and Z isomers have similar uorescence lifetimes. The dierence is more pronounced in viscous solvents.
Fluorescence lifetime trends for p CK − recorded waterethylene-glycol mixtures are shown in Figure S8: Wavelength dependence of observed uorescence lifetime for p CK − in water excited at 400 nm (at T = 300 K). Wavelengths given in the inset are the monitoring positions over the emission band. Figure S9: Example uorescence upconversion data, ts and residuals for p CK − excited at 400 nm (at T = 300 K). (a) methanol single-exponential t, (b) octanol single-exponential t, (c) methanol double-exponential t, and (d) octanol double-exponential t. Figure S10 (tted lifetimes are tabulated in Table S2). Dependence of the tted excited state-lifetime for p CK − on the solvent polarity (dielectric constant) is summarized in Figure S11. Following an earlier study, 11 there are linear correlations between ln (k f ) and 1 ε for alcohol solvents (ow cell) and waterethylene-glycol mixtures (static cell), and are related to the radii and charges or dipole moments of the reactant and excited state barrier. 26 However, quantitative information is dicult to extract because the viscosity dependence Figure S10: Fluorescence decay ts for p CK − in waterethylene-glycol mixtures. Percentages indicate vol% of water at T = 300 K. Fitted lifetimes are given in Table S2. is large. For the waterethylene-glycol mixtures, it is reasonable to assume preferential solvation of the chromophore (e.g. rst coordination sphere) by water molecules, leading to a degree of non-linearity in the correlation in Figure S11b. In the framework of electrostatic solute-solvent interactions, a negative slope in the correlations in Figure S11 show that excited state lifetime increases with solvent polarity and suggest that a more polar solvent decreases the barrier height by stabilizing the product state. This trend has been interpreted as consistent with a substantial charge shift accompanying excitation. 11,27,28 Figure S11: Dependence of k f ≈ 1 τ 2 for p CK − on solvent polarity, ϵ (dielectric constant), at T = 300 K: (a) a series of alcohols using a ow cell, (b) waterethylene-glycol mixtures using a static cell. Table S2: Fluorescence lifetimes (in ps) of pCK − in various solvents and solvent mixtures measured using static (s) and ow (f) cells (at T = 300 K), and obtained from single-(τ 0 ) and double-exponential tting (τ 1 and τ 2 ). τ 1 is limited by experiment crosscorrelation (55 fs). Percentages indicate vol% of water in the waterethylene-glycol solutions. ± indicates the tted uncertainty. EtGly is ethylene glycol.